Through covalent immobilization of template and surface imprinting, smart molecularly imprinted polymer nanospheres were developed for selective separation of the glycoprotein ovalbumin (OB). First, a boronic acid group-bearing poly (methyl methacrylate) (b-PMMA) nanosphere was synthesized directly at the high temperature of 70 degrees C. The b-PMMA nanosphere could pre-immobilize the template OB on its surface by forming reversible covalent bonds. Then the precipitation polymerization of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm) readily occurred on the b-PMMA nanosphere as a core at room temperature, leading to the formation of core-shell molecular imprinting nanosphere. The experiments showed the rebinding affinity of the imprinted nanospheres was thermo and pH-dependent. The resulting imprinted nanospheres showed high adsorption capacity and good specific recognition behavior toward the template molecule, and no obvious reusability deterioration was observed. Most notably, the imprinted nanospheres reached saturated adsorption within 20 min, indicating faster rebinding kinetics. In addition, the imprinted nanospheres were successfully applied to selectively separate the target OB from an egg white sample. (C) 2013 Elsevier B.V. All rights reserved.